Phosphorodiamidate Morpholino Oligomers: The Resistance of Our Organism

As its stated in the journal, The Future of Antibiotics and Resistance, the World Economic Forum (WEF) stated that “arguably the greatest risk … to human health comes in the form of antibiotic-resistant bacteria. We live in a bacterial world where we will never be able to stay ahead of the mutation curve. A test of our resilience is how far behind the curve we allow ourselves to fall.” Understanding that antibiotics have become an arising problem in the medical community for a few decades now has lead scientists wondering how we can stay above the curve we always fall behind. However, in order to resolve the problem, an understanding of antibiotics and researching new methods of treatment were needed.

Antibiotics are a form of medicine produced by bacteria that have been around for almost a century, combating bacterial diseases by inhibiting their growth and reducing death rates more and more each year. Antibiotics were first introduced in the 1940’s, but discovered in 1928 by Alexander Fleming. He discovered antibiotics when sorting through some contaminated petri dishes and observing newly formed colonies. He then isolated the colonies and their research from that was published to the world.

However, it was at Oxford where Howard Florey, Ernst Chain, and their colleagues examined its benefits for killing bacteria in the body, creating penicillin. Afterwards, when other antibiotics were introduced to the medical community, scientists further explained how they work. Initially, there are several different types of groups of antibiotics, beta lactam, macrolides, and others. These antibiotics enter the system, either not allowing the bacteria to construct their cell walls, dissolving their membranes, or even affecting protein building machinery in the bacteria. However, although they did not target human cells, the antibiotics did target good bacteria as well. This lead to a high emergence of vulnerability for elder persons who have been exposed to the antibiotic for a long period of time, which proceeded to result in infections.

When first introduced, there were many factors affecting the strength and success rates of antibiotics. A few common causes of failure having been high fevers, untreatable infectious diseases, or problems with incorrect spectrums. Although it was expected that resistance would occur at some point, no one expected it to happen so quickly. Nevertheless, doctors, pharmacists, and researchers continued using them and tried finding new ways to also improve the antibiotics. However, poor sanitation aided in the spreading of bacteria and funds for research was running low. So, about 60 years ago to be exact, signs of antibiotic resistance began to blossom into an even bigger problem. Even with the constant research on how to improve these antibiotics, more and more antibiotics eventually came to a point where they were at a plateau for some individuals.

The plateau meant antibiotic resistance was occurring. The first signs of resistance appeared in hospitals with high use of antibiotics and continued to spread from there on. It was found that with the population having been introduced to them for a long duration of time, complications of resistance in human cells were arising. As resistance increased in antibiotics, it also increased through other forms of treatment as well. Many people saw this as a threat, for it resulted in high amounts of uncured rates, which soon produced high amounts of deaths as well.

After the discovery of antibiotic resistance, scientists began examining cells to see what other plausible causes of the resistance would be. The examination of resistance began within the cells of the body, for all it takes is one bacteria surviving to multiply and pass down its resistant traits. This examination then transitioned to the idea of overuse in antibiotics, even though any amount of usage could potentially result in resistance, it is very unlikely. Initially, what happens is high consumptions of said antibiotic allowed the bacteria to alter their gene expression and become immune to treatment. However, in order for an overuse in treatment to occur, patients needed to be prescribed the antibiotics. So, why was this occurring?

Certain individuals, doctors especially, were beginning to have confusion between when to prescribe antibiotics because it became such a regular, normal thing. Although, most of the time, the confusion came from inadequate diagnostics. These two factors are what cause such high, unnecessary use of treatment. To be more specific on how large the scale of over usage is worldwide, nearly 30 percent, or 47 million prescriptions are unnecessarily prescribed amongst doctors’ offices today. Besides that, more overuse of antibiotics used in agricultural feed is also believed to assist in antibiotic resistance. The reason behind this begins with antibiotic resistance in livestock receiving nearly 80 percent of antibiotics annually. Our consumption of the newly developed resisting bacteria transfers them into our body for multiplication and so on.

Today, in the united states alone, there are over two million cases of infection from antibiotic resistance. These infections lead to about 23,000 deaths per year. Sadly, any sign of a resistance requires double recovery time in the hospital, with high costs resulted from testing new methods of treatment that could possibly rid the disease from the host. So, the idea of ceasing antibiotic use came into play.

However, to continue a life without treatment of antibiotics would mean that everything a person does or possibly contracts could lead death. Even small things, depending on how one would obtain it, a cut could become as life threatening as any incurable disease. Luckily, one of these new methods of treatment that is not as costly as others involves antisense mechanisms that are introduced to the patient through injections. The key purpose of an antisense mechanism is to use antisense drugs that bind to a target RNA forming two strands of molecules that do not allow the RNA from functioning or creating disease- causing proteins.

One of these antisense mechanisms used are peptide conjugated phosphorodiamidate morpholino oligomers, or PMOs for short. Phosphorodiamidate morpholino oligomers are uncharged DNA analogues that are capable of impeding on bacterial growth through gene- specific antisense mechanisms, or in other words, altering gene expression.

They can maintain thermal stability in cells as well, which is important because antibiotic resistant bacteria have processing temperatures. Through thermal stability, temperature of the application can remain constant, resisting change and altering the bacteria. Its molecular structure consisting of an RNA backbone and phosphorodiamidate groups have allowed them to have been used to target areas of open RNA structure for muscular dystrophy, alter sequences for the protection of ebola, and other methods of treatment as well.

When PMOs were discovered around 1993, researchers were ecstatic, for they had found a new alternative to antibiotics that were exceptionally low in production cost as compared to other treatments. They discovered the PMOs while observing a tat protein trans-activating an HIV promoter after crossing membranes, which identified a peptide responsible for the transactivation. After discovery, the peptide identified as cell-penetrating peptides, or CPP, was evaluated with other proteins and compounds such as PMOs.

First tested on rats, the variety of resistance on enzymes present in biological fluids, meant that PMOs were ideal for testing in vivo and in vetro applications. After further testing of PMOs, it was found that they were especially successful in inhibiting viral gene expression and replication in said animal models. The process of how PMOs were administered to the rats, or any in vivo/vetro application for any experiment involving the mechanism occurred in two different ways. PMOs can be administered through a syringe as either intravenous (IV) or intraperitoneal (IP) injections.

Intravenous injections are given through the veins and intraperitoneal are injected into somewhere in the body cavity. Afterwards, CPPs carry the PMO throughout the body where it can begin replication or target harmful bacteria. Based off of the design of the PMO, during this process, they can bind to pre-mRNA and alter gene splicing, which would soon result in an addition or exclusion of specific genetic fragments once mRNA has matured.

However, delivery was not ideal, so researchers covalently conjugated PMOs to the CPPs. Later on, in other areas of research, it was found that the length and target distance areas of PMOs affected the resistant area significantly. PMOs that were closer to the acceptor site were more active and longer PMOs were better at inducing exon skipping. At this point, the PMOs would also begin to alter the RNA sequence of the bacteria. Once altered, the antibiotic resistant bacteria would slowly die out, thus proving to researchers that PMOs were successful in curing said bacteria. So, once FDA approved, PMOs were introduced to the public.